In vitro scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole derivative drugs

This study was undertaken to evaluate the scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) by several nonsteroidal anti-inflammatory drugs (NSAIDs), namely indole derivatives (indomethacin, acemetacin, etodolac), pyrrole derivatives (tolmetin and ketorolac), and an oxazole derivative (oxaprozin). The inhibition of prostaglandin synthesis constitutes the primary mechanism of the anti-inflammatory action of these drugs. Nevertheless, it has been suggested that the anti-inflammatory activity of NSAIDs may be also partly due to their ability to scavenge ROS and RNS and to inhibit the respiratory burst of neutrophils triggered by various activator agents. Thus, the scavenging activity of these NSAIDs was evaluated against an array of ROS (O(2)(-), HO, HOCl, and ROO) and RNS (NO and ONOO(-)) using noncellular in vitro systems. The results obtained demonstrated that tolmetin, ketorolac, and oxaprozin were not active against O(2)(-), while acemetacin, indomethacin, and etodolac exhibited concentration-dependent effects. Oxaprozin was also the least active scavenger for HO, among all the tested NSAIDs shown to be active. The scavenging effect for HOCl was not observed for any of the tested NSAIDs. The ROO was effectively scavenged by etodolac, with the other tested NSAIDs being much less active. NO and ONOO(-) were scavenged by all the tested NSAIDs. These effects may strongly contribute to the anti-inflammatory therapy benefits that may be attained with some of the studied NSAIDs.     

Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

It has long been known that singlet oxygen ((1)O2) is generated during inflammatory processes. Once formed in substantial amounts, (1)O2 may have an important role in mediating the destruction of infectious agents during host defense. On the other hand, (1)O2 is capable of damaging almost all biological molecules and is particularly genotoxic, which gives a special relevance to the scavenging of this ROS throughout anti-inflammatory treatments. Considering that the use of non-steroidal anti-inflammatory drugs (NSAIDs) constitutes a first approach in the treatment of persistent inflammatory processes (due to their ability to inhibit cyclooxygenase), a putative scavenging activity of NSAIDs for (1)O2 would also represent a significant component of their therapeutic effect. The aim of the present study was to evaluate the scavenging activity for (1)O2 by several chemical families of NSAIDs. The results suggested that the pyrazole derivatives (dipyrone and aminopyrine) are, by far, the most potent scavengers of (1)O2 (much more potent compared to the other tested NSAIDs), displaying IC(50)-values in the low micromolar range. There was a lack of activity for most of the arylpropionic acid derivatives tested, with only naproxen and indoprofen displaying residual activities, as for the oxazole derivative, oxaprozin. On the other hand, the pyrrole derivatives (tolmetin and ketorolac), the indolacetic acid derivatives (indomethacin, and etodolac), as well as sulindac and its metabolites (sulindac sulfide and sulindac sulfone) displayed scavenging activity in the high micromolar range. Thus, the scavenging effect observed for dipyrone and aminopyrine will almost certainly contribute to their healing effect in the treatment of prolonged or chronic inflammation, while that of the other studied NSAIDs may have a lower contribution, though these assumptions still require further in vivo validation.     

An exploratory theoretical elucidation on the peroxyl-radical-scavenging mechanism and structure-activity relationship of nonsteroidal anti-inflammatory drugs

The peroxyl-radical-scavenging mechanism of some nonsteroidal anti-inflammatory drugs (NSAIDs), namely tolmetin, ketorolac, indomethacin, acemetacin, and oxaprozin, is clarified by combined density functional theory (DFT) calculations. It is revealed that H-atom-abstraction rather than electron transfer reaction is involved in the radical-scavenging process of these NSAIDs in polar aqueous solution. This seems contrary to the common viewpoint that the latter is predominant in polar media. The calculated results also show that H-atom at C(beta) or C(gamma) position is readily to be abstracted, and the lowest C-H bond dissociation enthalpy (BDE) can qualitatively account for the activity difference for the five NSAIDs.     

The metabolism of sulindac enhances its scavenging activity against reactive oxygen and nitrogen species

Sulindac is a sulfoxide prodrug that, in vivo, is converted to the metabolites sulindac sulfide and sulindac sulfone. It is therapeutically used as an anti-inflammatory and analgesic in the symptomatic treatment of acute and chronic rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In addition to its anti-inflammatory properties, sulindac and its metabolites have been shown to have an important role in the prevention of colonic carcinogenesis. Although the inhibition of prostaglandin synthesis constitutes the primary mechanism of action of sulindac, it is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammation and cancer. Thus, the aim of this study was to evaluate the scavenging activity of sulindac and its sulfone and sulfide metabolites for an array of ROS (HO*, O2(*-), and HOCl) and RNS (*NO and ONOO-) using in vitro systems. The results we obtained demonstrate that the metabolism of sulindac increases its scavenging activity for all RNS and ROS studied, notably with regard to the scavenging of HOCl. These effects may strongly contribute to the anti-inflammatory and anticarcinogenic efficacy that has been shown for sulindac.     

A Fas-associated death domain protein-dependent mechanism mediates the apoptotic action of non-steroidal anti-inflammatory drugs in the human leukemic Jurkat cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.     

Sulindac inhibits activation of the NF-kappaB pathway.

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IkappaB kinase beta (IKKbeta) that is required to activate the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-kappaB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKbeta kinase activity. Concentrations of sulindac that inhibit IKKbeta activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-kappaB pathway.     

Non-steroidal anti-inflammatory agents, tolmetin and sulindac, inhibit liver tryptophan 2,3-dioxygenase activity and alter brain neurotransmitter levels

Hepatic tryptophan 2,3-dioxygenase (TDO) is one of the rate-limiting enzymes in tryptophan catabolism and plays an important role in regulating the physiological flux of tryptophan into relevant metabolic pathways. In this study, we determined the effect of the non-steroidal anti-inflammatory agents, tolmetin and sulindac, on rat liver TDO activity and the subsequent changes in the hippocampal and striatal neurotransmitter levels. The amount of melatonin produced by the pineal gland was also measured using high performance liquid chromatography (HPLC). Treatment of rats with tolmetin or sulindac (5 mg/kg/bd for 5 days) significantly inhibited liver TDO activity. The results show that whilst tolmetin and sulindac increase serotonin levels in the hippocampus, these agents also significantly reduce dopamine levels in the striatum. Tolmetin, but not sulindac, increased the amount of melatonin produced by the pineal gland. The results of this study suggest that whilst tolmetin and sulindac may be beneficial for patients suffering from depression, these agents also have the potential to induce adverse effects in patients suffering with neurological disorders such as Parkinson's disease.     

Scavenging of reactive oxygen species by some nonsteroidal anti-inflammatory drugs and fenofibrate

Ketoprofen and tolmetin are widely used nonsteroidal anti-inflammatory drugs, whereas fenofibrate belongs to a family of hypolipidemic drugs used in the prevention of cardiovascular diseases. The aim of this study was to assess effect of these drugs on reactions generating reactive oxygen species (ROS). The following generators of ROS were used: 18-crown-6/KO(2) dissolved in DMSO as a source of superoxide radical (O(.-)(2), the Fenton-like reaction (Cu/H(2)O(2)) for hydroxyl radical (HO(.)), 2,2'-azobis (2-amidino-propane) dichloride (AAPH) as peroxyl radical (ROO(.)) generator, and a mixture of alkaline aqueous H(2)O(2) and acetonitrile for singlet oxygen ((1)O(2)). Measurements were done using chemiluminescence, fluorescence, and spin-trapping with 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy (ESR), and a deoxyribose assay based on the spectrophotometry. The results obtained demonstrated that all tested drugs were active against O(.-)(2). There was a clear ranking of drug inhibition effects on chemiluminescence from the O(.-)(2) system: ketoprofen > tolmetin > fenofibrate. The examined compounds inhibited the HO(.)-dependent deoxyribose degradation and scavenged the ROO(.) concentration dependently with an order of potencies similar to that of the superoxide radical system. Hence, these results indicate that the studied drugs show broad ROS scavenging property and, as a consequence, might decrease tissue damage due to the ROS and thus to contribute to anti-inflammatory therapy.     

Up-regulation of GADD45α expression by NSAIDs leads to apoptotic and necrotic colon cancer cell deaths

Growth arrest and DNA damage inducible 45 alpha (GADD45α) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45α in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45α mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45α is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45α expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45α in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.     

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin gamma1 in human glioblastoma cells

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Efficacy and specificity of non-steroidal anti-inflammatory drugs for the inhibition of cytokine-stimulated prostaglandin E(2) secretion by amnion-derived WISH cells

Prostaglandin H synthase-2 (PGHS-II) specific inhibitors have been proposed as a potential treatment in the prevention of preterm birth. We examined the efficacy of PGHS inhibitors on basal and cytokine-stimulated prostaglandin (PG) production by the amnion-like WISH cell line. WISH cells were treated with interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha in the presence or absence of indomethacin, etodolac, 5,5-dimethy-3-(3-fluorophenyl)-4-(4-methlysulphonyl) phenyl-2 (5H)-furanone (DFU) or nimesulide (1.6-1000 nM) for 16 h. PG production was then measured using radioimmunoassay. Nimesulide and DFU were the most selective non-steroidal anti-inflammatory drugs (NSAIDs) of IL-beta-stimulated PG production in these studies with an a IC(50)(basal)/IC(50)(stimulated) ratio of, respectively, 142.2 and 113.8, followed by etodolac (25.3) and indomethacin (2.2). Similar results were obtained when cells were stimulated with TNF-alpha. The results of this study suggest that PGHS-II-selective NSAIDs may be effective in the prevention of cytokine-driven amnion PG production associated with preterm labour.     

Indomethacin and ibuprofen induce Hsc70 nuclear localization and activation of the heat shock response in HeLa cells

It has been established that non-steroidal anti-inflammatory drugs (NSAIDs), such as sodium salicylate, sulindac, ibuprofen, and indomethacin, induce anti-inflammatory and anti-proliferative effects independent of cyclooxygenase. These cyclooxygenase-independent pharmacodynamic effects appear to regulate several signaling pathways involving proliferation, apoptosis, and heat shock response. However, the mechanisms of these actions remain an area of ongoing investigation. Hsc70 is a cytoplasmic chaperone protein involved in folding and trafficking of client proteins to different subcellular compartments, plays roles in signal transduction and apoptosis processes, and translocates to the nucleus following exposure to heat shock. Since NSAIDs induce some aspects of the heat shock response, we hypothesized that they may also induce Hsc70 nuclear translocation. Western immunoblotting and indirect cellular immunofluorescence showed that indomethacin and ibuprofen induce Hsc70 nuclear translocation at concentrations previously shown to induce HSF DNA-binding activity. Chemical inhibition of both p38(MAPK) and Erk42/44 had no effect on localization patterns. In addition, while indomethacin has been shown to behave as an oxidative stressor, the radical scavenging agent, N-acetyl cysteine, did not inhibit nuclear translocation. These results indicate that induction of the heat shock response by NSAIDs occurs at concentrations fivefold greater than those required to inhibit cyclooxygenase activity, suggesting a cyclooxygenase-independent mechanism, and in the presence or absence of kinase inhibitors and a free radical scavenger, suggesting independence of Erk42/44 or p38(MAPK) activities and intracellular oxidoreductive state.     

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.     

Nonsteroidal anti-inflammatory drugs inhibit gastric peroxidase activity

The peroxidase activity of the mitochondrial fraction of rat gastric mucosa was inhibited with various nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro. Indomethacin was found to be more effective than phenylbutazone (PB) or acetylsalicylic acid (ASA). Mouse gastric peroxidase was also very sensitive to indomethacin inhibition. Indomethacin has no significant effect on submaxillary gland peroxidase activity of either of the species studied. Purified rat gastric peroxidase activity was inhibited 75% with 0.15 mM indomethacin showing half-maximal inhibition at 0.04 mM. The inhibition could be withdrawn by increasing the concentration of iodide but not by H2O2. NSAIDs inhibit gastric peroxidase activity more effectively at acid pH (pH 5.2) than at neutral pH. Spectral studies showed a bathochromic shift of the Soret band of the enzyme with indomethacin indicating its interaction at or near the heme part of the enzyme.     

Spectroscopic behavior of copper complexes of nonsteroidal anti-inflammatory drugs

The proposed curative properties of Cu-based nonsteroidal anti-inflammatory drugs (NSAIDs) have led to the development of numerous Cu(II) complexes of NSAIDs with enhanced anti-inflammatory activity. Crystalline complexes, Cu(II)-NSAID (ibuprofen, naproxen, tolmetin, and diclofenac), with a carboxylic function have been studied by means of infrared and Raman spectroscopy. All NSAIDs bind the metal through the carboxylate group. On the basis of the comparison between the wavenumber of the COO(-) group vibrations and Delta nu (nu(asimm)COO(-) - nu(simm)COO(-)) between Na salts and Cu(II) complexes, conclusions on the probable structure of the complexes have been drawn. The spectroscopic data support the formation of dimeric [Cu(2)L(4)(H(2)O)(2)] complexes in which the COO(-) group behaves as a bridging bidentate ligand. The low wavenumber region of the Raman spectrum provided information on Cu-O and Cu-Cu bonds in the complexes. Thermogravimetric results gave further support to the vibrational data.     

Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp²³-Lys²⁸ salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly³³, a residue involved in Met³⁵ oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.     

Cutting edge: agonistic effect of indomethacin on a prostaglandin D2 receptor, CRTH2.

Indomethacin is a widely used nonsteroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting cyclooxygenases or activating peroxisome proliferator-activated receptors. In this study, we present evidence demonstrating that the novel PGD(2) receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is another functional target for indomethacin. Indomethacin induced Ca(2+) mobilization in CRTH2-transfected K562 cells at submicromolar concentrations (approximate EC(50), 50 nM) in a G(alphai)-dependent manner as PGD(2) did. Other nonsteroidal anti-inflammatory drugs (aspirin, sulindac, diclofenac, and acemetacin) had no such effect even at micromolar concentrations. In chemotaxis assay, three CRTH2-expressing cell types, Th2 cells, eosinophils, and basophils, were all significantly attracted by indomethacin (EC(50), 50-500 nM) as well as by PGD(2) (EC(50), 2-20 nM), and the effects of indomethacin were blocked by anti-CRTH2 mAb. These results suggest the involvement of CRTH2 in mediating some of therapeutic and/or unwanted side effects of indomethacin, independently of cyclooxygenases and peroxisome proliferator-activated receptors.